An early diagnosis of asthma in young children by using non-invasive biomarkers of oxidative stress/airway inflammation, and early lung function measurements

Asthma is a disease characterised by chronic airway inflammation. The
prevalence of asthma in children in Western industrialised countries varies
between 5-30%, and has increased in the past two decades. There are large
problems with the diagnosis of asthma in young children. About 70% of children
with recurrent asthma-like symptoms is symptom-free at 6 years and does not has
asthma. The other 30% has asthma. Currently, an early and reliable diagnosis
of asthma in young children is not possible with the conventional diagnostic
measures. However, such a reliable diagnosis is important for the correct
treatment of young children with respiratory symptoms. An effective therapy of
asthma by means of anti-inflammatory treatment with inhaled corticosteroids is
available. This treatment has a beneficial influence on airway inflammation,
respiratory symptoms, asthma exacerbations, quality of life, lung function, and
may reduce irreversible damage (*airway remodelling*) to the airways. It is
also known that inhaled corticosteroids are not very effective in children with
transient wheezing, and therefore treatment of these children is unnecessary,
and leads to preventable costs and side-effects. Therefore, an early diagnosis
will prevent undertreatment of true asthmatics and overtreatment of transient
wheezers. This is of great importance for both infants and parents. In the past
few years, relatively new lung function tests became available, and has been
applied in young children. So far, measurements of oxidative stress/airway
inflammation do not play a part in the diagnosis of asthma. It is probable that
inflammatory biomarkers in exhaled air, and venous blood can reliably indicate
an asthma diagnosis.

This research proposal tests the hypothesis that an early asthma diagnosis is
possible by using non-invasive biomarkers of oxidative stress/airway
inflammation in exhaled air, venous blood and assessments of lung function
(airway resistance by means of the interrupter technique).

A standardised questionnaire on respiratory symptoms (ISAAC) will be completed
in 1500 children aged 2-3 years in general practice (from the Registration
Network of Family Practices of the University of Maastricht) and a birth cohort
study (KOALA study). From the results of the ISAAC questionnaire, a
prospective, matched, case control study is started of 200 children with
recurrent respiratory symptoms (= cases) and 50 control subjects (=controls)
with no or minor respiratory symptoms. At 6 years, a reliable, definite
diagnosis of asthma is made (primary outcome measure) on basis of lung function
assessments (maximal expiratory flow-volume curves before and after a
bronchodilator, bronchial hyperresponsiveness) and current respiratory
symptoms. At 2-3 years, repeated measurements of important predictors are
performed:1) nitrogen monoxide (NO) in exhaled air; 2) a profile of
inflammatory biomarkers in exhaled air by means of gas chromatography
time-of-flight mass spectrometer (GC-TOF-MS); 3) airway resistance before and
after a bronchodilator by means of the interrupter technique (Rint); 4) the
improvement in inflammatory biomarkers and airway resistance during a 2-month
treatment with an inhaled corticosteroid.
At the start (2- 3 years) and at the end (6 years) of the study 8 ml venous
blood, a swab and a faeces sample will be sampled for 1) white blood cell
count, differentiation, number of eosinophils; 2) total and specific IgE; 3)
cytokine profiles; 4) the presence and function of regulatory T-cells; 5)
markers of oxidative stress; 6) gene expression of markers of oxidative stress
and inflammation; 7) polymorfism of relevant genes; 9) infection serology

A trial with inhaled corticosteroids is part of the diagnosis phase of this
study. Children in the experimental group will enter a controlled crossover
trial with inhaled corticosteroids. This trial consists of a treatment period
of 2 month with inhaled corticosteroid therapy (beclomethasone 2 times 100
microgram a day via the Airochamber ®), and 2-month period with placebo. Half
the children start with inhaled corticosteroids, half start with placebo. All
other anti-inflammatory medication will be stopped 4 weeks before the trial.

This research will last three to four years (dependent of the age of the child
at entry). In the first year we will investigate the effect of a 2 month
treatment with inhaled corticosteroids on lung function and inflammatory
biomarkers in exhaled air. Measurements will take place at the start of the
study, after 2 months and after 4 months. In the second and third year,
measurements will take place once a year. At 6 years, a reliable, definite
diagnosis of asthma is made.

Each visit at the hospital contains of 5 measurements:
1. A questionnaire will be taken to assess e.g. medication, respiratory symptoms
2. NO in exhaled air will be measured according to the standards of the ERS/ATS
. Exhaled air is collected via a face mask, covering the mouth, which is
connected to a non-rebreathing valve that allows inspiration of NO-free air
from a NO-inert reservoir to avoid contamination by ambient NO.
3. Exhaled breath condensate will be collected during tidal breathing, in a
cooled tube for 10 minutes while children are wearing a nose-clib. During this
test children can watch cartoons.
4. During tidal breathing, expired air will be collected in a 3-litre inert
bag by means of a 2-way valve system.
5. Measurements of airway resistance will be performed by means of a simple
lungfunction measurement.

At the start (2- 3 years) and at the end (6 years) of the study 8 ml venous
blood, a swab will be sampled. A faeces sample will be collected